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Methods in Pharmacology and Toxicology Molecular Histopathology and Tissue Biomarkers in Drug and Diagnostic Development Steven J. Potts David A. Eberhard Keith A. Wharton, Jr. Editors M E T H O D S I N P H A R M A C O L O G Y A N D T O X I C O L O G Y Series Editor Y. James Kang Department of Medicine University of Louisville School of Medicine Prospect, Kentucky, USA For further volumes: http://www.springer.com/series/7653 Molecular Histopathology and Tissue Biomarkers in Drug and Diagnostic Development Edited by Steven J. Potts Flagship Biosciences, LLC, Westminster, CO, USA David A. Eberhard University of North Carolina, Chapel Hill, NC, USA Keith A. Wharton, Jr. Novartis Institutes for BioMedical Research, Cambridge, MA, USA Editors Steven J. Potts Flagship Biosciences, LLC Westminster, CO, USA Keith A. Wharton, Jr. Novartis Institutes for BioMedical Research Cambridge, MA, USA ISSN 1557-2153 ISSN 1940-6053 (electronic) Methods in Pharmacology and Toxicology ISBN 978-1-4939-2680-0 ISBN 978-1-4939-2681-7 (eBook) DOI 10.1007/978-1-4939-2681-7 Library of Congress Control Number: 2015939268 Springer New York Heidelberg Dordrecht London # Springer Science+Business Media New York 2015 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to thematerial contained herein or for any errors or omissions that may have beenmade. Printed on acid-free paper Humana Press is a brand of Springer Springer Science+Business Media LLC New York is part of Springer Science+Business Media (www.springer.com) David A. Eberhard University of North Carolina Chapel Hill, NC, USA Dedication Chris Callahan, M.D., Ph.D. This book is dedicated to the memory of our dear friend and esteemed colleague, Chris Callahan, M.D., Ph.D., who suffered an untimely death from a progressive brain tumor in 2011. Already recognized as a leader early in his career, Chris held the position of Scientist and Investigative Pathologist at Genentech/Roche at the time of his passing at the young age of 46. Chris’s professional contributions pervade this book’s themes of molecular histopathology and tissue-based biomarkers, and several of its contributors trained with or worked alongside Chris at various stages of his career. More importantly, Chris represented a generation of visionary physician-scientists who began their training in the 1980s, the last “pre-genome” decade of human history, with the belief that the discovery of molecules and pathways governing normal development would provide key insights into human disease. While now considered dogma, at that time only scattered yet tantalizing hints existed to indicate that alterations of these same handful of pathways, deeply conserved during our evolution, caused (and were druggable targets of) diverse human diseases. Chris completed his B.S. at Brown University in 1987, and earned his M.D. and Ph.D. degrees as a graduate student in John Thomas’s lab at UC San Diego and Salk Institute performing groundbreaking work in Drosophila neurobiology. His accomplishments include the molecular cloning of derailed, a receptor tyrosine kinase crucial for axon guidance [1]. Recognizing the central role of pathology in identifying molecular and cellular mechanisms of disease, Chris moved to Stanford University to pursue residency training in anatomic pathology, ultimately serving as Attending Physician and Acting Assistant Professor of Pathology while engaged in postdoctoral research in Dermatology with Tony Oro (also a former fellow M.D./Ph.D. student with Chris at UC San Diego). Extending Tony’s postdoctoral work with Matthew Scott that implicated aberrant Hedge- hog signaling in the most common human tumor, basal cell carcinoma [4], Chris went on to discover a critical role for a Hedgehog pathway target gene Mtss1 (Missing in Metasta- sis) in the regulation of signaling and cancer progression [3]. Tragically, Chris’s cancer diagnosis came at a most inopportune time—just as he sought to start his own lab. Following aggressive surgery and chemotherapy, he returned to the bench within days to continue his passion. By this time, Hedgehog pathway v inhibitors were being developed for oncology indications, and Chris felt the best opportu- nity to apply his skills and talents to directly benefit cancer patients was to continue his career as a pathologist in biopharma. Chris joined Genentech, supporting drug develop- ment projects while directing several research projects aimed at understanding the mechan- isms of Hedgehog pathway activation and therapy resistance in human cancer. Chris was a key contributor to the R&D team efforts that led to the FDA approval of vismodegib, the first-in-class Hedgehog pathway inhibitor for clinical use in advanced basal cell carcinoma. The several hats Chris wore in these efforts included basic research and medical scientist, translational biomarker and companion diagnostics R&D investigator, and pathology advisor to the development team. Chris investigated cancer mechanisms to the end of his life, with his final senior author paper on the role of Hedgehog signaling in tumor-stroma interactions published in the Proceedings of the National Academy of Sciences just a few months before his death [4]. Chris’s approach to life inspired those around him, as these quotes from two of his Genentech colleagues attest: (I have) never met anyone with more selfless dedication, engagement, focus, and commitment to his work and family than Chris. Chris loved his role as a pathologist at Genentech and immersed himself in it 100 %. He knew that his work was helping to transform the lives of patients, and felt fortunate that he could do so by engaging in professional activities he loved most—basic hypothesis-driven research and scientific collaboration. Chris knew, more clearly than most of us do, that his time with his family, his friends, and his work was limited. He relished that time, and he shared it generously with others. Chris had a selfless, collegial enthusiasm for his work. He was absolutely committed to his colleagues and to the projects he supported; he would do everything he could to maximize the chances for their success. He had a great appreciation of, and loved sharing subtle details of biology—not to advertise his brilliance, but because he trusted you would find them as satisfying and wonderful as he did. When he dropped off his sons at school in the morning he would tell them, ‘Have fun, learn a lot, and be kind.’ He fully modeled that advice. Beyond describing Chris to a tee, these reminiscences illustrate three qualities of a successful anatomic pathologist in biopharma that emerge as themes throughout this book: a focus on the biology of disease, a passionate curiosity, and a collaborative mindset. Although new insights about disease emerge daily, and opportunities for new discoveries have never been greaterthan the present time, some things haven’t changed: A century and half ago, Rudolf Virchow, founder of cellular pathology, said “If we would serve science, we must extend her limits, not only as far as our own knowledge is concerned, but in the estimation of others.” [5]. Chris exemplified this Virchowian ideal. Comprehending the biology of disease requires integration of knowledge from diverse disciplines, of which we concede the histopathology “stock in trade” of fixed and stained tissue is only one part. As a role model, Chris excelled at the challenges faced by anatomic pathologists embedded in drug and diagnostic industries, chief among them to bridge the power (and limitations) of histopathology methods and knowledge with those from a growing number of technology-driven disciplines, including genomics, protein biochem- istry, quantitative image analysis, and in vivo imaging of cells to whole animals. Chris saw firsthand that delivering a diagnostic test or new therapy to patients requires diverse skills, far beyond what any one individual could possibly master. Whether performed in industry or research institutes, drug development requires coordinated efforts by multidisciplinary teams, and so the pathologist on the team must persuasively communicate with members from diverse backgrounds and viewpoints in order to foster collaboration, and ultimately, progress. Rare talents like Chris, with curious and creative minds, able to integrate emerging data and knowledge across disciplines, are poised to see old problems in new vi ways and develop novel, important hypotheses that demand investigation. As Virchow said, the pathologist must push the science—“. . .extend her limits. . .”—for all to see. Chris fearlessly pursued multidisciplinary investigations in fruit flies, mice, and human systems in order to understand core biologies and their alterations in human disease. We don’t yet know if flies or mice will benefit from the fruits of Chris’s research, but humanity has already benefited, and for that we are most grateful. Chris and his wife Andrea are the proud parents of two boys, Nathan and Ryan. Acknowledgements Special thanks to Tony Oro, Cary Austin, and UCSD, Stanford, and Genentech colleagues for their contributions to this dedication. Cambridge, MA, USA Keith A. Wharton, Jr. Chapel Hill, NC, USA David A. Eberhard References 1. Callahan CA, Bonkovsky JL, Scully AL, Thomas JB (1996) derailed is required for muscle attachment site selection in Drosophila. Development 122(9):2761–2767 2. Oro AE, Higgins KM, Hu Z, Bonifas JM, Epstein EH, Jr., Scott MP (1997) Basal cell carcinomas in mice overexpressing sonic hedgehog. Science 276(5313):817–821 3. Callahan CA, Ofstad T, Horng L, Wang JK, Zhen HH, Coulombe PA, Oro AE (2004) MIM/BEG4, a Sonic hedgehog-responsive gene that potentiates Gli-dependent transcription. Genes Dev 18 (22):2724-2729. doi:10.1101/gad.1221804 4. Chen W, Tang T, Eastham-Anderson J, Dunlap D, Alicke B, Nannini M, Gould S, Yauch R, Modrusan Z, DuPree KJ, Darbonne WC, Plowman G, de Sauvage FJ, Callahan CA (2011) Canonical hedgehog signaling augments tumor angiogenesis by induction of VEGF-A in stromal perivascular cells. Proc Natl Acad Sci U S A 108 (23):9589-9594. doi:10.1073/pnas.1017945108 5. Virchow R (1858) Cellular pathology (trans: Chance F). Edwards Brothers, Inc., Ann Arbor, MI vii Preface I’ve just sucked one year of your life away. . . What did this do to you? Tell me. And remember, this is for posterity so be honest. How do you feel? –Count Rugen, antagonist in the 1987 movie The Princess Bride In the movie The Princess Bride, the hero, Westley, has just been subjected to The Machine, a torture device that sucks years of life out of the victim. Like Westley, who cries andmoans in pain in response to Count Rugen’s query, anyone embarking on, or reflecting upon, a multi-year project knows the pathologic feeling of time spent on a lengthy and complex project, whether it is a book, a drug, or a film. Feature films aspiring for blockbuster status can consume $100 million or more in production costs and 3 years just to get to production stage—all to entertain people for a mere 2 hours. Yet this amount of money pales in comparison to the economic realities of producing a new therapeutic that might address an unmet medical need for thousands or even millions of people. By most measures, developing a new drug in 2015 costs at least ten times more than a blockbuster movie. Three years in production is feature film fiction compared to the industry average of ~14 years for drug development. The feature film and the pharmaceutical industries face similar challenges: years between the initial idea and a revenue-generating product, huge multifaceted teams, millions of dollars invested in multiple projects, only a few of which succeed, and the hope of the occasional blockbuster that must finance the failures of the rest. At each phase in drug development, from early discovery through IND (Initial New Drug Application) to NDA (New Drug Application), the promise of efficacy is balanced against the penalty of toxicity. While there are many ways that efficacy and toxicity can be evaluated in animals and in people, the highest concentration of information relevant to many diseases remains the lesional tissue sample, microscopic examination of which pro- vides a foundation to understand disease and the effect of therapy. Increasingly, whole microscopic slide imaging is used, providing at least an order of magnitude higher resolu- tion of cellular context than current noninvasive in vivo radiological imaging techniques. However, microscopic data requires many players to extract its maximum value: histology (preparing the tissue sample) and pathology (interpreting the tissue sample), in addition to experts in disease-specific biology. Tissue-based studies help to understand how candidate therapies act in animals and humans, and this work is often performed by small biotech companies, large pharmaceutical companies, academic medical centers, commercial refer- ence laboratories, and government entities. Each actor has a critical role to play in the process and in the development of the final product. We anticipate those who will most benefit from reading this book will be embedded in government-sponsored academic research, diagnostics, or biopharmaceuticals, but we have strived to make the chapters accessible and interesting to a wide audience. Due to shrinking government budgets for basic research, more academic researchers are responding to grant announcements and pharmaceutical partnerships that drive them deeper into drug devel- opment. With the growth of companion diagnostics, experts in disease diagnostics will find useful information in this volume about co-development of diagnostic and therapeutic products, though the nature and timelines of the diagnostic industry are very different from those of drug development, creating some unanticipated but, on deeper ix consideration, not so surprising challenges. Our analogy to the film industry provides caution to those entering pharmaceutical drug development: While the biology underlying drug development may be familiar—and thus appear simple—to those outside the bio- pharma industry, one cannot overstate the complexities of drug development. One should approach the study of the biopharma industry, and one important part of it—tissue histopathology—with the same caution one might approach completely unfamiliar terri- tory: with curiosity and respect for lessons learned by experience. Scientists from the diagnostics industry are forewarned: while pharma and diagnostics have shared biology, they are as dissimilar as are the pharma and moviemaking industries. We often celebrate 2 hours of entertainment more than we give pause to acknowledge medicines that positively impact humanlives. Recent progress in hepatitis C, cystic fibrosis, and tumor immunology has been nothing short of astounding. Our industry can be the best at times and the worst at times, but for many of us there is no more satisfying endeavor than the opportunity to design therapeutics that have the potential to save and improve lives. It is our heartfelt belief that the biopharmaceutical industry makes positive and lasting contributions to humanity. With the human genome completed just over a decade ago, comparative genomics studies have revealed an array of druggable targets whose manipu- lation is at the root of most therapy development programs today. In the not-so-distant future, we are poised to witness dramatic improvements in the treatment of a myriad of severe and debilitating diseases including infectious diseases, intractable and largely incur- able cancers, as well as autoimmune and genetic diseases. Histopathology is central to this effort, yet it is often relegated to a checkbox activity that is not given proper scrutiny or thought. Our authors and editorial team, consisting of experts in histopathology, have written from the trenches of diagnostic practice and pharmaceutical drug development, aiming to educate pharmaceutical and academic scientists how to best use tissue in drug development. Most pathologists and histologists are, by their very nature, humble and not oriented to marketing their wares. This book aims to help make their contributions to drug develop- ment better understood as well as to identify best practices and new applications for their trade. The book’s dedication highlights the contribution made by an exemplary individual—a pathologist no longer in our midst—whose example continues to motivate us. Audience This book is intended for three audiences. First and foremost, it is written for all scientists and managers in the Biopharma industry who must interact directly or indirectly with tissue samples but whose primary training did not include pathology or other skills of tissue interpretation; second, for pathology professionals and tissue scientists who will find some of the examples of applications of their trade in drug development by their peers and colleagues helpful; and third, for the many academic groups funded by government entities to become more engaged in all stages of drug development. x Preface Information Content of a Tissue Biopsy Both clinicians and the general public expect the pathologist’s interpretation to be the “gold standard” of disease diagnosis—the absolute truth. “What were the path results?” “Do I have cancer or not? What kind?” In most cases, pathological interpretation of a relevant tissue sample is the final arbiter of truth. In both efficacy and toxicology studies, there is much information to be gleaned from local tissue environment and context, with the spatial and temporal characteristics of the cells in their organ preserved. While we strive to link cell and tissue-level resolution with complex datasets that derive from -omics analyses such as next-generation DNA and RNA sequencing, many forget that immuno- histochemistry has provided single cell analysis of protein distribution—albeit a single protein at a same time—for decades. Technology is always creating new approaches to interrogate tissue samples, both within biopharma and in academia, but for a technology to be implemented in clinical practice, it requires confirmation of utility and definition of its limits and context that often comes years after the technology has lost its newness. But the expectation that magically circulating in the blood is information content that will replace the efficacy and toxicology information content of a tissue biopsy is a fantasy of Hollywood proportions. Many people, even within the biopharma industry, are not aware that every organ of every animal used in a GLP (Good Laboratory Practice) toxicology study must be examined under the microscope by one or more veterinary pathologists. The FDA and other regulatory agencies remain aware of the importance of the tissue microenvironment to drug development. How Important Is Histopathology in Drug Development? How do we assign an economic value to tissue analysis in drug development? One approach is to estimate the number of slides generated or read per year, which then can be converted to dollars. This can be estimated in several different ways, working from the number of pathologists who engage in testing or the number of drugs that require testing of tissue exposed to them. There are approximately 600 veterinary pathologists who work primarily in pharmaceutical research, and probably another ~40 M.D. pathologists exclu- sively working in pharmaceutical research. The vast majority of slides utilized in the pharmaceutical industry are in support of GLP toxicological pathology studies for IND submissions. Previous studies have estimated that on average one pathologist supports $1B of drug product development [1]. We can assume each pathologist reviews ~10,000 slides a year (~50 slides a day � ~200 working days a year) and yields an estimate of nearly ~7 million glass slides evaluated each year worldwide in drug development. However, this number does not account for non-boarded pathologists and scientists who review slides, so it may likely be closer to ten million glass slides per year. Even with a rough estimate of $150 per glass slide for creation (histology) and reading (pathology), this equates to $1.5 billion spent annually on histopathology within the pharmaceutical industry. Preface xi Organization and Goals While much of the focus of pathology in the biopharmaceutical industry is on oncology programs, there is growing recognition of the value of pathology outside of oncologic disease. Consequently, the chapters have been deliberately selected to include other disease areas, including chapters addressing nonalcoholic steatohepatitis, arthritis, celiac disease, myeloproliferative disorders, neurology, and wound healing. As part of a methods series, this volume is designed to provide practical wisdom and examples that others can follow and apply as part of drug development. Some chapters are case studies in specific techni- ques or disease areas where tissue biopsies can be utilized effectively, while others are literature reviews, and still others are a summary of the authors’ decades of collective experience with tissue in an area of drug development. While a great deal of pathology-related efforts in drug development are by necessity geared toward formal toxicologic evaluation under GLP requirements, the editors have deliberately not focused on toxicologic pathology as a discipline. This is certainly not reflective of the relative importance of safety testing, but was rather for two other reasons: First, toxicologic pathology in drug development has been comprehensively covered for over a decade in a regularly updated excellent handbook for practitioners [2] as well as in specialty journals. Second, the pace of change in toxicologic pathology is slow in compari- son to other more technology-driven aspects of pathology commonly used in drug development. The IND approval process allowing first-in-human studies is a highly regu- lated endeavor, stipulated by GLP standards, that can take on a life (and a career) of its own. Of necessity, innovation occurs slowly—and often reactively—in this field. This book, which focuses on pursuits within industry that are variably termed “experi- mental” or “investigative” pathology, or “translational medicine,” thus deals more with efficacy studies that bridge from early stage discovery to formal clinical trials. An introduc- tion to the field of anatomic pathology and its application to the biopharma industry is first provided, based on the author’s experience both in industry and through teaching medical students. This is followedby a personal narrative by a leading biopharma pathologist on the nuances of communicating pathology results to non-pathologist colleagues, and then by a chapter on planning and outsourcing histopathology-based investigations in clinical trials. Two leading experts in inflammatory disease then provide a specific example of how histopathology can be leveraged to better understand rheumatoid arthritis. The second section focuses on applications of tissue image analysis–whole microscopic slide imaging and computer algorithms to quantitatively measure what the pathologist qualitatively observes. The chapters cover a variety of disease areas and concepts including angiogenesis, hepatic fibrosis, and celiac disease, providing a glimpse of future applications of digital pathology. The third section discusses molecular histopathology, divided into in situ hybridization (mRNA and DNA), sequencing, and genomics. The reader will find state-of-the-art reviews with methodology on in situ hybridization as well as next-generation sequencing of tissue samples. The fourth section covers companion diagnostics. The first two chapters describe preanalytic variables and then the adaptation of HER2 IHC scoring systems commonly used in breast cancer to gastrointestinal tumors. Three chapters then discuss the develop- ment of companion diagnostics from an industry standpoint, the relationship between the xii Preface reference lab, diagnostic partner and the pharma client, and regulatory aspects of medical device submissions. A success story of a multianalyte IHC companion diagnostic is then presented. Finally, two biostatisticians discuss statistical approaches to cut-point analysis in digital pathology and applications in IHC companion diagnostics. We hope that this volume will serve to inform and enlighten both tissue-focused and non-tissue-focused drug development scientists about better use and interpretation of the multidimensional data contained in a tissue biopsy. The hunt for new therapies remains one of the most exciting and meaningful pursuits in the twenty-first century, and so the evaluation of a tissue biopsy remains a central and challenging part of that pursuit. Westminster, CO, USA Steven J. Potts Cambridge, MA, USA Keith A. Wharton, Jr. Chapel Hill, NC, USA David A. Eberhard References 1. Potts SJ, Young GD, Voelker FA (2010) The role and impact of quantitative discovery pathology. Drug Discov Today 15(21–22): 943-50 2. Haschek WM, Rousseaux CG, Wallig MA (2002) Handbook of toxicologic pathology, Vol 1. Academic, San Francisco Preface xiii Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix Dedication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xvii Histopathology: A Canvas and Landscape of Disease in Drug and Diagnostic Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Keith A. Wharton Jr A Field Guide to Homo morphologicus for Biomedical Scientists, Or How to Convey an Understanding of Pathology to Scientists in a Biopharma Enterprise . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 Humphrey Gardner Outsourcing Tissue Histopathology Investigations in Support of Clinical Trials for Novel Therapeutics: Considerations and Perspectives . . . . . . . . 43 Keith A. Wharton Jr., Benjamin H. Lee, Pierre Moulin, Dale Mongeon, Rainer Hillenbrand, Arkady Gusev, Bin Ye, and Xiaoyu Jiang Histopathology in Mouse Models of Rheumatoid Arthritis . . . . . . . . . . . . . . . . . . . . . 65 Patrick Caplazi and Lauri Diehl Markers Used for Visualization and Quantification of Blood and Lymphatic Vessels. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 Mohamed E. Salama, David A. Eberhard, and Steven J. Potts Practical Approaches to Microvessel Analysis: Hotspots, Microvessel Density, and Vessel Proximity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87 Steven J. Potts, David A. Eberhard, and Mohamed E. Salama Quantitative Histopathology and Alternative Approaches to Assessment of Fibrosis for Drug Development in Hepatitis C and Nonalcoholic Steatohepatitis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 Steven J. Potts and Johanna K. DiStefano Stereology and Computer-Based Image Analysis Quantifies Heterogeneity and Improves Reproducibility for Grading Reticulin in Myeloproliferative Neoplasms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117 Mohamed E. Salama, Erik Hagendorn, Sherrie L. Perkins, Jeff L. Kutok, A. Etman, Josef T. Prchal, and Steven J. Potts Image Analysis Tools for Quantification of Spinal Motor Neuron Subtype Identities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127 Mirza Peljto and Hynek Wichterle Development of a Tissue Image Analysis Algorithm for Celiac Drug Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141 Erik Hagendorn, Christa Whitney-Miller, Aaron Huber, and Steven J. Potts Quantitative Histopathology for Evaluation of In Vivo Biocompatibility Associated with Biomedical Implants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153 Robert B. Diller, Robert G. Audet, and Robert S. Kellar xv Quantitative Histomorphometry and Quantitative Polymerase Chain Reaction (PCR) as Assessment Tools for Product Development . . . . . . . . . . . . . . . . . 163 Robert G. Audet, Robert B. Diller, and Robert S. Kellar Measuring the Messenger: RNA Histology in Formalin-Fixed Tissues. . . . . . . . . . . . 175 Steven J. Potts, Mirza Peljto, Mahipal Suraneni, and Joseph S. Krueger Algorithm-Driven Image Analysis Solutions for RNA ISH Quantification in Human Clinical Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183 Mirza Peljto, Joseph S. Krueger, Nicholas D. Landis, G. David Young, Steven J. Potts, and Holger Lange Solid Tissue-Based DNA Analysis by FISH in Research and Molecular Diagnostics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191 Marcus Otte Preanalytic Considerations for Molecular Genomic Analyses of Tissue. . . . . . . . . . . . 203 Maureen Cronin Next-Generation Sequencing (NGS) in Anatomic Pathology Discovery and Practice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219 Matthew J. McGinniss, David A. Eberhard, and Keith A. Wharton Jr The Impact of Pre-analytic Variables on Tissue Quality from Clinical Samples Collected in a Routine Clinical Setting: Implications for Diagnostic Evaluation, Drug Discovery, and Translational Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259 David G. Hicks Adapting HER2 Testing for a Different Organ: New Wine in Old Wineskins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271 Michael D. Lunt and Christa L. Whitney-Miller Tissue-Based Companion Diagnostics: Development of IHC Assays from an Industry Perspective . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . 281 Miu Chau and Jon Askaa Development of Tissue-Based Companion Diagnostics: The Relationship Between the Pharmaceutical Company, Diagnostic Partner, and the Biomarker Laboratory . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305 Mark Kockx, Stefanie de Schepper, and Christopher Ung Navigating Regulatory Approval for Tissue-Based Companion Diagnostics . . . . . . . 325 Joseph S. Krueger, Holger Lange, G. David Young, and Steven J. Potts Implementing a Multi-analyte Immunohistochemistry Panel into a Drug Development Program . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345 Carla Heise, Pierre Brousset, Tommy Fu, David A. Eberhard, Graham W. Slack, Camille Laurent, and Randy D. Gascoyne Cutpoint Methods in Digital Pathology and Companion Diagnostics . . . . . . . . . . . . 359 Joshua C. Black, Mahipal V. Suraneni, and Steven J. Potts Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373 xvi Contents Contributors JON ASKAA � Medical Prognosis Institute, Hørsholm, Denmark ROBERT G. AUDET � Development Engineering Sciences LLC, Flagstaff, AZ, USA JOSHUA C. BLACK � Flagship Biosciences, Westminster, CO, USA PIERRE BROUSSET � Department of Pathology, CHU Toulouse-Purpan, Toulouse, France PATRICK CAPLAZI � Department of Research Pathology, Genentech, Inc., South San Francisco, CA, USA MIU CHAU � Genentech, Inc., South San Francisco, CA, USA MAUREEN CRONIN � Strategic Information Management, Celgene Corporation, San Francisco, CA, USA LAURI DIEHL � Department of Research Pathology, Genentech, Inc., South San Francisco, CA, USA ROBERT B. DILLER � Development Engineering Sciences LLC, Flagstaff, AZ, USA; Department of Biological Sciences, Center for Bioengineering Innovation, Northern Arizona University, Flagstaff, AZ, USA JOHANNA K. DISTEFANO � TGEN, Phoenix, AZ, USA DAVID A. EBERHARD � Department of Pathology and Laboratory Medicine, Department of Pharmacology and Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC, USA; Laboratory Corporation of America (LabCorp), Research Triangle Park, NC, USA A. ETMAN � University of Utah & ARUP Laboratories, Salt Lake City, UT, USA TOMMY FU � Celgene Corporation, Summit, NJ, USA HUMPHREY GARDNER � Translational Medicine, Early Clinical Development, AstraZeneca R&D, Waltham, MA, USA RANDY D. GASCOYNE � Department of Pathology and the Center for Lymphoid Cancer, Organization British Columbia Cancer Agency, Vancouver, BC, Canada ARKADY GUSEV � Biomarker Development, Translational Medicine, Novartis Institutes for BioMedical Research, Cambridge, MA, USA ERIK HAGENDORN � Flagship Biosciences, Westminster, CO, USA CARLA HEISE � Celgene Corporation, Summit, NJ, USA DAVID G. HICKS � Surgical Pathology Unit, Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, Rochester, NY, USA RAINER HILLENBRAND � Biomarker Development, Translational Medicine, Novartis Institutes for BioMedical Research, Basel, Switzerland AARON HUBER � School of Medicine and Dentistry, University of Rochester, Rochester, NY, USA XIAOYU JIANG � Biomarker Development, Translational Medicine, Novartis Institutes for BioMedical Research, Cambridge, MA, USA ROBERT S. KELLAR � Development Engineering Sciences LLC, Flagstaff, AZ, USA; Department of Biological Sciences, Center for Bioengineering Innovation, Northern Arizona University, Flagstaff, AZ, USA; Department of Mechanical Engineering, Center for Bioengineering Innovation, Northern Arizona University, Flagstaff, AZ, USA MARK KOCKX � HistoGeneX, Antwerp, Belgium JOSEPH S. KRUEGER � Flagship Biosciences, Westminster, CO, USA xvii JEFF L. KUTOK � Infinity Pharmaceuticals, Inc., Cambridge, MA, USA NICHOLAS D. LANDIS � Flagship Biosciences, Westminster, CO, USA HOLGER LANGE � Flagship Biosciences, Westminster, CO, USA CAMILLE LAURENT � Department of Pathology, CHU Toulouse-Purpan, Toulouse, France BENJAMIN H. LEE � Oncology Translational Medicine/Oncology Business Unit, Novartis Institutes for BioMedical Research, Cambridge, MA, USA MICHAEL D. LUNT � School of Medicine and Dentistry, University of Rochester, Rochester, NY, USA MATTHEW J. MCGINNISS � Medical Laboratory, Genoptix, a Novartis Company, Carlsbad, CA, USA DALE MONGEON � Biomarker Development, Translational Medicine, Novartis Institutes for BioMedical Research, Cambridge, MA, USA PIERRE MOULIN � Discovery and Investigative Pathology, Preclinical Safety, Novartis Institutes for BioMedical Research, Basel, Switzerland MARCUS OTTE � Oridis Biomarkers, Graz, Austria MIRZA PELJTO � Flagship Biosciences, Westminster, CO, USA SHERRIE L. PERKINS � University of Utah & ARUP Laboratories, Salt Lake City, UT, USA STEVEN J. POTTS � Flagship Biosciences, Westminster, CO, USA JOSEF T. PRCHAL � University of Utah & ARUP Laboratories, Salt Lake City, UT, USA MOHAMED E. SALAMA � Department of Pathology, ARUP Reference Lab Research Institute, University of Utah, Salt Lake City, UT, USA STEFANIE DE SCHEPPER � Immunohistochemistry, HistoGeneX, Antwerp, Belgium GRAHAM W. SLACK � Department of Pathology and the Center for Lymphoid Cancer, Organization British Columbia Cancer Agency, Vancouver, BC, Canada MAHIPAL V. SURANENI � Flagship Biosciences, Boulder, CO, USA; Flagship Biosciences, Westminster, CO, USA CHRISTOPHER UNG � HistoGeneX, Antwerp, Belgium KEITH A. WHARTON JR. � Discovery and Investigative Pathology, Preclinical Safety, Novartis Institutes for BioMedical Research, Cambridge, MA, USA CHRISTA L. WHITNEY-MILLER � School of Medicine and Dentistry, University of Rochester, Rochester, NY, USA HYNEK WICHTERLE � Department of Pathology, Columbia University, New York, NY, USA; Department of Neurology, Columbia University, New York, NY, USA; Department of Neuroscience, Columbia University, New York, NY, USA BIN YE � Biomarker Development, Translational Medicine, Novartis Institutes for BioMedical Research, Cambridge, MA, USA; Beijing Shenogen Biomedical Co., Beijing, China G. DAVID YOUNG � Flagship Biosciences, Westminster, CO, USA xviii Contributors Methods in Pharmacology and Toxicology (2015): 1–26 DOI 10.1007/7653_2014_33 © Springer Science+Business Media New York 2014 Published online: 22 January 2015 Histopathology: A Canvas and Landscape of Disease in Drug and Diagnostic Development Keith A. Wharton Jr. Abstract The aims of diagnostic and therapeutic development are to accurately diagnose and cure disease, respectively. For the past century and a half, histopathology—the microscopic examination of cells and tissues—has been considered a “gold standard” for the diagnosis of many diseases. As an introduction to this volume on molecular histopathology and tissue biomarkers in drug and diagnostic development, I explore the relationship between histopathology and the nature of disease itself. A lack of agreement on the meaning of “disease” has led to widespread and indiscriminate use of the term. Here, I propose that the term “disease” be reserved for conditions where there exists some knowledge of alterations in cells or their products that participate in cause-effect relationships in lesional (diseased) tissue. This is a definition that simultaneously lends legitimacy to the term’s use while enabling revision and testing of hypotheses based on rapidly emerging scientific knowledge. With this perspective, histopathology, as a preferred means to visualize and depict the cellular events that constitute disease as it impacts tissue structure and function, will remain essential to develop new diagnostic tests and targeted therapies for the foreseeable future. Key words Disease, Histopathology, Biomarker, Lesion, Diagnosis, Illness, Condition,Disorder, Pathogenesis, Feedback, Crosstalk, Clinical trial, Diagnostic test, Drug development 1 Introduction If thought corrupts language, language can also corrupt thought. —George Orwell I rob banks because that’s where the money is. —Willie Sutton The concept of “disease” is central to understanding health in all forms of life as well as for optimal delivery of health care. All human cultures have words or symbols that represent disease or related ideas. From our earliest experiences, we develop an understanding of its general nature, but the term is increasingly adapted to suit circumstances at hand. Diagnostic criteria, a set of features that define each disease, range from simple to complex. For some well- understood diseases, diagnosis is clear-cut, relying on defined and measurable criteria. For poorly understood or controversial 1 diseases, observation and professional judgment play a major role, creating opportunities for uncertainty, bias, and misdiagnosis. Health care providersmake treatment decisions and bill for reimbur- sements based on an incomplete and evolving understanding of many diseases. Perusal of http://www.clinicaltrials.gov/, the USA FDA’sWeb site thatdiscloses the essentials of all humanclinical trials, reveals that enrollment criteria and response variables (i.e., trial end- points) formanydiseases consist of features learned from themedical history, or of clinical observations andmeasurements with uncertain relevance to the disease processes under investigation. To the afflicted individual, having a particular disease can alter self-identity and behavior, and can impart social stigma and/or advantage. Disease, frankly, is a big deal. Given that shared goals of biomedical research, the biopharmaceutical industry, and the health care indus- try as a whole are accurate diagnosis and treatment of disease, as an introduction to this timely book devoted to molecular histopathol- ogy and tissue biomarkers in drug and diagnostic development, it seems reasonable to first consider the nature of disease itself. Now, early in the twenty-first century, humanity finds itself squarely in the middle of the molecular era of disease, full of optimism that, from our recently sequenced genomes, cures for our myriad diseases are imminent. Investigations from diverse fields of biomedicine have revealed over the past 50 or so years the molecular underpinnings, or at least a framework, for thousands of hitherto mysterious and sometimes misclassified diseases— spanning from the rare and precisely defined to the common and heterogeneous. For example, there are at least 456 distinct genetic diseases that affect the skeletal system [1], a majority for which causative gene mutations have been identified. We have learned that the culprits in many human (and animal) diseases are corrupted versions of vital processes deeply conserved in our evolutionary history—alterations in specific proteins, protein complexes, macro- molecular structures, and organelles that comprise molecular com- munication pathways with developmental, homeostatic, and adaptive functions. In some cases, we know which cells misbehave and how, and we can replicate key features of the disease in wild- type or genetically altered animals. Since organisms are composed of massive networks of interconnected, compartmentalized molec- ular pathways, the task that remains for each disease, in each patient, is to understand the dynamic, physical nature of each pathway alteration and then devise strategies to oppose the abnor- mal network of behaviors that promote disease without compromising functions of the remaining pathways required for life. 2 Keith A. Wharton Jr. 2 What Is Histopathology? Histopathology is the microscopic visualization of tissue to describe how cellular and tissue morphology is altered by manifestations of disease (http://en.wikipedia.org/wiki/Histopathology). It is dis- tinct from techniques that use (and destroy) tissue as a raw material for nucleic acid sequencing or bioassays to measure specific mole- cules or their concentrations, each of which separate the data generated from its endogenous tissue context. Histopathology thus provides a visual, descriptive picture of cells and tissues, fixed or frozen in time, in their natural context—a landscape of sorts. When viewed using the microscopic techniques of histopathology, normal tissue might resemble a well-ordered city when viewed from the sky, whereas tissue from a destructive autoimmune disease might look like a war zone, and a cancer might kindle images of prisoners escaping Alcatraz or Godzilla tipping skyscrapers. Histo- pathology is central to patient care, whether to discern normal tissue from benign or malignant disease, to ascertain surgical tissue margins, or to investigate cause of death. Among all diagnostic assays, histopathology remains unparalleled in its ability to discrim- inate among a seemingly infinite variety of disease states, especially in the presence of a much greater excess of normal tissue. For example, in an appropriate biopsy, a trained pathologist can diag- nose prostate cancer by recognizing less than ten malignant (cancerous) cells among thousands of nonmalignant cells. Histopa- thology is also a crucial tool to assess efficacy and possible safety hazards of candidate human therapies in preclinical (i.e., animal) studies. Despite the regulatory requirement for histopathology- based interpretation of tissues in animal toxicology studies, collect- ing tissues as part of human clinical trials remains a rare practice, except in oncology trials. (A later chapter in this book explores issues to consider when incorporating tissue and histopathology- based measurements in human clinical trials.) Anatomic pathologists are the sages of histopathology. They are mostly physicians (M.D., D.O., or equivalent) or veterinarians (D.V.M. or equivalent) who have completed one or more anatomic pathology fellowships and often have research experience in inves- tigative pathology or related scientific disciplines. They train over years and decades to render interpretations and diagnoses based on observable and, to an outsider, sometimes arcane criteria. Despite the fact that Pathology, translated from its Greek origins, literally means the study of suffering, pathology emerged as a medical specialty, in part, to provide an objective diagnosis of disease, unbi- ased by patient presentation or a physician’s subjective (but often crucial) knowledge about a patient’s history. While a computer, IBM’s Watson, can win the TV game show Jeopardy, no computer is yet capable of replacing a diagnostic anatomic pathologist. Histopathology: A Canvas and Landscape of Disease in Drug and Diagnostic Development 3 In this book, the editors and contributors adopt a far broader view of histopathology, to include any discipline or technique that enhances our ability to visualize and quantify the three- dimensional, physical nature of normal and diseased tissue, includ- ing advanced techniques in microscopy and cell biology, image segmentation and quantitation, omics-scale profiling of various molecular species, and “big data” analysis. Thus histopathology is not a static or archaic art based only on routine stains of tissue sections, but rather highly technical and continually evolving. These ancillary technologies thus add more, and often critical, detail to the landscape that is each disease. Modern histopathology is a firmly molecular pursuit, encompassing the detection of specific proteins by immunohistochemistry (IHC) and nucleic acids by in situ hybridization (ISH). The editors espouse the view that data generated from techniques that destroy tissue must be interpreted in the context of methods such as histopathology that preserve it, in order to best understand the manifestations of each diseasein tissue and in the patient. Modern histopathology thus requires patholo- gists to collaborate with a wide variety of scientists and engineers who generate complementary, often deeper insights from diseased tissue, in order to create a coherent working model of each disease process. 3 What Is Disease? Historical concepts of disease arose independently in diverse civili- zations, including causation by external factors (e.g., gods, demons, spells, miasmas) and imbalances in internal qualities (e.g., the four humors). Largely incorrect in their primitive forms, each view has elements of truth: disease can be initiated by external factors (e.g., infectious agents, toxin exposure) and can originate or manifest as imbalances in internal qualities (e.g., persistent hormone produc- tion or accumulation of toxic metabolites, mutations, or rogue cells). Historical review of disease conceptions across cultures is outside the scope of this chapter, but several sources exist [2–4]. Clinical acumen and technology jointly drive the discovery and diagnosis of disease. Many diseases are—or were, at some point in history—named for the person, usually a physician, who first noticed a nonrandom aggregation of clinical or histopathological features in their patients; Alois Alzheimer and James Parkinson are two whose namesakes persist, though many names of diseases, proper and otherwise, have since been discarded in favor of more descriptive designations. The light microscope revealed fine details of healthy and diseased tissues in the mid-nineteenth century, revolutionizing disease diagnosis, classification, and nomenclature. Thousands of discrete disease entities are recognized by the Inter- national Classification of Diseases, ninth revision (ICD-9), a list 4 Keith A. Wharton Jr. subject to revision every few years based on emerging knowledge and expert consensus. Recognize, however, that such lists are made largely for the purposes of counting and billing, and their compo- sition usually lags behind the most current thinking about disease. Today, a panoply of omics techniques (genomics, proteomics, etc.) and in vivo imaging modalities give unprecedented insight into the complexity of disease as it affects our bodies; these technologies challenge prevailing notions of disease, force continual reassess- ment of naming conventions, and can allow earlier, more accurate diagnosis and monitoring of disease. For example, diagnosis of Alzheimer’s dementia, in the past requiring an autopsy, can now be made premortem with some certainty by combining cognitive testing with imaging and cerebrospinal fluid biomarker analysis [5]. Massively parallel (next generation) sequencing, the subject of another chapter in this book, stands to revolutionize disease diag- nosis, classification, and monitoring in the next decade. Principles of systems biology are being applied to datasets derived from diseased tissue or fluids in an attempt to better classify, understand, and treat disease (e.g., ref. 6). Such studies create compelling and often testable hypotheses. However, the enormous sizes of datasets in comparison to the typically small numbers of diseased samples used to generate them have raised concern that resulting predictive models will be overfitted to the diseased sample set and thus not applicable to the population as a whole. Many consider “disease” a more specific concept than simply the absence of health, and defining it, in general and for each specific case, is not a trivial exercise. One classic definition of disease, articulated by Sir John Russell Reynolds in 1866, is: “any condition of the organism which limits life in either its power, enjoy- ment, or duration” [7]. Merriam Webster (www.m-w.com) defines disease as, “a condition of the living body or of one of its parts that impairs normal functioning and is typically manifested by distin- guishing signs and symptoms.” (Recall that symptoms are experienced by the patient, such as insomnia or pain, whereas signs are observed by the physician during physical examination; e.g., Courvoisier’s sign is a palpable gallbladder in a patient with obstructive jaundice). Wikipedia (c. 2014) offers a wider, more inclusive definition: “. . .any condition that causes pain, dysfunction, distress, social pro- blems, or death to the person afflicted, or similar problems for those in contact with the person. In this broader sense, it sometimes includes injuries, disabilities, disorders, syndromes, infections, isolated symp- toms, deviant behaviors, and atypical variations of structure and function.” Unfortunately, here inclusion breeds imprecision, although given the mutable nature of Wikipedia the definition might be changed by the time you read this chapter! Such definitions of disease, both classic and modern, are inade- quate tools for thinking about and communicating modern con- cepts of disease. Too often language, entrenched by historical Histopathology: A Canvas and Landscape of Disease in Drug and Diagnostic Development 5 accidents of discovery, shapes the boundaries of our thought, when thought, shaped by scientific discovery, should enhance the accu- racy and precision of language that describes the products of our thought. These observations suggest a new working definition of disease is needed—one that not only reflects scientific principles upon which experts can agree, but that also facilitates thinking about new ways to treat or cure disease. This (or any) attempt to create a single umbrella definition for disease might be regarded as futile, but one hopes the effort helps to focus scrutiny on disease- relevant events. 4 Three Concepts Related to Disease Disease can be considered distinct from illness, which m-w.com defines as “unhealthy in body or mind,” but commonly refers a patient’s subjective experience of disease. Given that one can feel ill without having a known disease or have a severe disease with no ill feelings (e.g., early stage cancer), it is reasonable to assume that a defined disease need not be a prerequisite for illness, or at least temporarily ill feelings. Treating symptoms of disease, illness, and suffering are laudable goals, but the view of disease proposed here, absent knowledge about the disease process itself, might not inform the best way to manage illness. (Note that alternate definitions of “illness” are nearly synonymous with disease). Disease is distinct from a medical condition, which implies deviation from normalcy but is not necessarily an abnormality. For example, pregnancy is a temporary condition, but not an abnormal one. Disease is also distinct from a disorder, a term related to disease but with the implication that much less is known about the nature of what’s wrong. Disorder is often used to describe intrinsic abnormalities of brain function. For example, the Diag- nostic and Statistical Manual of Mental Disorders (DSM), currently in its fifth revision, is an ongoing but imperfect attempt to classify disorders of thought, feeling, or behavior. A few entities described in the current DSM qualify as a “disease” by the standards of this chapter (e.g., those conditions highly associated with mutations in genes that act in known pathways impacting specific regions of the brain), but their mysterious nature precludes classification by cri- teria more precise than those based on constellations of behavior or symptoms. Disorder is also used as a term that refers to a family of diseases or their manifestations, e.g., disorders of metabolism. Designation as “disease” remains controversial and is not with- out consequence. Ill feelings, a condition, or even normal physio- logical variation can be “medicalized” or “pathologized” into disease bymotivated parties, including scientists seeking grant fund- ing or biopharmaceutical companies seeking a new market niche. For example, thereis ongoing debate whether certain life-cycle- 6 Keith A. Wharton Jr. associated conditions are bona fide diseases or are simply conse- quences of aging that many in Western society refuse to accept. Companies that make products such as nutritional supplements not subject to FDA approval craft language to imply that their product is beneficial (“supports the health of. . .” is a commonly used phrase) for this or that organ (and by implication, will oppose disease of that organ) while required byUSA federal law to state that their “product is not intended to diagnose, treat, cure, or prevent any disease.” Carrying a diagnosis of a so-named disease has socioeco- nomic consequences. The American Medical Association’s recent declaration that obesity is a disease drew attention to its increasing prevalence and impact on society’s health [8], but at the same time affixed a label of questionable utility beyond possession of a “risk factor” for other diseases such as diabetes, hypertension, and myocardial infarction to millions of overweight people. Often, implicit in affixing the label of “disease” is absolution of blame for the affected individual’s state of health or actions resulting from it. Addiction, personality disorders, or willful criminal acts: are they diseases or not? Any debate about the nature of disease can quickly transcend science, reaching the realm of philosophy and raising the question: Do we control the disease or does the disease control us? Highlighting our pervasive inability to agree on what constitutes disease, an insightful New Yorker magazine article [9] quotes E.M. Jellinek’s mid-twentieth century work that advocated alcoholism as a disease: “A disease is what the medical profession recognizes as such.” Given the stakes, there is need for a working definition of disease that simultaneously lends credibility to use of the term, while enabling revision of disease concepts based on emerging scientific knowledge. I propose that bona fide “disease” be consid- ered an abnormality of the organism, caused by abnormal cells or their products, leading to cause-effect relationships in lesional tissue that compromise function in a societal context or attenuate normal lifespan. Through visualization of diseased cells and tissues, histo- pathology becomes both the canvas and the landscape upon which disease unfolds. 5 Organic Disease vs. Functional Disease In line with the proposed definition of disease, the medical profes- sion has long distinguished between organic and functional disease. Organic disease can cause a measurable physiological change, and thus changes in molecules, cells, tissues, or organs that give rise to the disease (or for which such changes are a consequence of the disease) can be identified and often measured with a diagnostic test. Functional diseases, sometimes called functional symptoms or dis- orders, can manifest as signs, symptoms, or somehow altered Histopathology: A Canvas and Landscape of Disease in Drug and Diagnostic Development 7 function (e.g., pain, fatigue, or other form of suffering) without apparent alterations in known tests. As science progresses, many functional disorders will join the ranks of organic diseases. For example, “inflammatory bowel disease” encompasses several organic diseases of the bowel in which alterations in tissue structure documented by histopathology generally correlate with the severity of clinical symptoms, whereas “functional bowel disease” (a.k.a. “irritable bowel syndrome”), despite sometimes equally debilitat- ing symptoms, is characterized by an absence of specific abnormali- ties by histopathology, but has been associated with clinical depression. The organic/functional distinction can become blurred in certain instances: although we do not know exactly which cells are altered, and how, in the brains of patients with schizophrenia or autism, few doubt their organic nature. Some disorders currently classified as functional are associated with psychological or psychi- atric conditions, raising the question of whether they are somatic (body) manifestations of an underlying psychiatric condition or disorder. Pathologists claim dominion over organic disease, leaving the more difficult questions of functional disease and its manage- ment to other specialists. A diagnosis of exclusion is one that is made after other causes are excluded: some diseases, like the multisystem granulomatous disease sarcoidosis, are clearly organic, whereas others not currently linked to definitive test results are classified as functional but may be no less a burden than organic disease to those afflicted. 6 Three More Concepts Related to Disease Three more concepts deserve mention before I further examine the nature of disease. A syndrome is a nonrandom association of several apparently unrelated features (signs, symptoms, test results) that suggest a common underlying cause or pathogenesis. Down syndrome, a constellation of developmental defects due to trisomy 21, and Acquired Immune Deficiency Syndrome (AIDS), caused by HIV infection-induced immune deficiency, are classic examples. Pathognomonic describes the almost certain presence of a disease (or disorder) when a particular sign or symptom, or a combination thereof, is present, or with a particular diagnostic test result. In statistical terms, this means that the “positive predictive value” (the percentage of patients with a feature such as a positive test result that have thedisease) of a particular sign, symptom,or test result is 100%. Two skin diseases illustrate these two points. Cutaneous xanthomas (small skin papules or nodules composed predomi- nantly of macrophages stuffed with cholesterol) are pathogno- monic for hyperlipoproteinemia; i.e., all individuals with xanthomas have some sort of alteration in lipoprotein metabolism. 8 Keith A. Wharton Jr. Dozens to hundreds of cutaneous basal cell carcinomas in sun- exposed areas on a young adult are pathognomonic for Gorlin’s basal cell nevus syndrome, caused by a heterozygous, germ line mutation in the PTCH1 gene [10]. But, how specific the relation- ship is between the pathognomonic feature and the disease varies with the definition and cause of each disease: not all individuals with hyperlipoproteinemias have xanthomas, and there are many differ- ent causes of hyperlipoproteinemia. In contrast, the relationship between PTCH1 mutation and Gorlin’s syndrome is obligate, in that there appears to be no other genes that when singly mutant give rise to such a specific, striking array of clinical manifestations. The third concept is forme fruste, which describes a very mild or atypical form of a disease. One example is discoid lupus, a variant of the autoimmune disease systemic lupus erythematosus (SLE) whose manifestations are restricted to skin. Sometimes, a forme fruste variant manifests in a distinct tissue or organ compared to the more typical or severe form of the disease. For example, cystic fibrosis, due to mutation in the CFTR channel protein, has severe, life-threatening manifestations in the lung and pancreas, but occa- sional patients with partial loss of function mutations in the same protein present with male infertility due to the requirement of CFTR for vas deferens function [11]. Hence, male infertility can be a forme fruste of cystic fibrosis. Since each of us harbors on the order of 400 defective genes in our genomes and inherits ~60 new mutations not present in our parents’ DNA [12, 13], widespread use of massively parallel sequencing technologies in future diagnos- tic tests (the subject of another chapter in this book) is likely to reveal combinations of sequence polymorphisms or otherwise cryp- tic gene mutations as factors contributing to “forme frustes” of many diseases. 7 Etiology/Chain of Causation The foundation of differential diagnosis—the processof determin- ing which among a list of potential diseases a patient might have based on their unique presentation—is classification of disease by apparent cause or etiology. Broad, descriptive categories have tradi- tionally been used to classify disease: these include neoplastic, infectious, genetic, vascular, and idiopathic—the latter signifying our ignorance of an underlying cause. Distinctions are made between congenital and acquired disease, and genetic, familial, and sporadic disease; these categories will not be further explored. Diseases, however (as defined), can be considered primary if they originate within the named organ, or secondary or even tertiary if causative factors originate from other organs. For example, primary hyperparathyroidism is caused by autonomous hypersecretion of parathyroid hormone (PTH) from the parathyroid gland (e.g., by a Histopathology: A Canvas and Landscape of Disease in Drug and Diagnostic Development 9 parathyroid cell-derived tumor or from nonmalignant parathyroid cells harboring a mutation that activates a signaling pathway, lead- ing to PTH hypersecretion). Since the function of PTH is to increase serum calcium by acting on a variety of target tissues (bone, kidney, intestine), secondary hyperparathyroidism is charac- terized by increased parathyroid secretion from the parathyroid gland, usually due to the body’s attempt to correct for low serum calcium. The manifestations of secondary hyperparathyroidism on target organs outside of the parathyroid gland are similar to those of primary hyperparathyroidism, yet the causes are very different. Sometimes, a presumptive or working diagnosis of disease is made if time is limiting and the consequences of missing a diagno- sis, and the appropriate intervention, are dire. For example, pneu- monia is commonly treated with medical therapy based on clinical history and symptoms, often confirmed with an X-ray, but without identification of a causative infectious agent. Even a definitive diag- nosis of a disease in a given patient should inspire further inquiry, sometimes urgently. For example, while iron deficiency anemia can be considered a disease—characterized by abnormal test results, signs, and symptoms—its presence beckons the search for a cause. Indeed, distinct, qualitative categories of disease, coupled with ordered (e.g., primary and secondary) events during disease pro- gression, implies that a sequence of discrete events, a so-called chain of causation, is a more accurate way to describe disease. Pathogenesis is a term used to describe the mechanism and sequence of key events typical of a disease or in a given patient with a disease. A related term, pathophysiology, emphasizes alterations in normal physiology caused by a disease. By analogy to a river, “upstream” and “downstream” refer to events that occur earlier or later in the chain of causation. Since correlation does not imply causation, we must be careful when describing a putative chain of disease causation that we do not assume one event causes another when both events are temporally distinct but are due to shared (or distinct), yet unknown, “upstream” causes. Nevertheless, in reference to a particular event in the disease (e.g., appearance of a sign or symptom, positive test result, evidence of disease progression, or even death), I borrow two terms from law, proximate cause and ultimate cause. A proximate cause is an occurrence or entity most closely responsible for the event of interest. For example, a proximate cause of most myocardial infarctions (heart attacks) is rupture of the fatty clot-forming material contained within an atherosclerotic plaque into the lumen of a coronary artery, resulting in thrombus formation, arterial lumen occlusion, and necrosis of myocardium in the artery’s territory. A proximate cause of the signs and symp- toms in many autoimmune diseases appears to be rare but crafty populations of autoreactive lymphocytes that escape elimination by the immune system, secreting proteins (e.g., TNF-alpha, IL17) 10 Keith A. Wharton Jr. that drive progression of disease, wreaking havoc on various tissues and organs. An ultimate cause (also known as root cause) is a further upstream event or entity “without which” the proximate cause or downstream events would not occur: Without atherosclerosis of coronary arteries, most myocardial infarcts would not occur, and without a mutation in the CFTR gene, cystic fibrosis (or its forme frustes) would not occur. Common ultimate causes are genetic mutations, environmental exposures, infectious agents, and inju- ries; without the inciting event, e.g., a mutation or exposure, the disease would likely not occur. For many diseases, ultimate causes remain elusive. While ordering events in disease pathogenesis can be a satisfy- ing exercise, it is often said that “life itself is a terminal disease,” implying that for every event, further upstream causes exist. For example, origins of atherosclerosis remain under intense investiga- tion, with evidence that genetic predisposition, environmental exposure (e.g., diet), and perhaps the interaction between inoppor- tune infections and the immune system, combine to create the lesions of atherosclerosis [14]. There is epidemiologic evidence in human, and experimental evidence in lower organisms, that paren- tal and even grandparental environment can affect health in adult- hood, possibly through epigenetic changes in DNA that are passed through the germ line [15]. Indeed, the term chain of causation implies a linear order of events in disease pathogenesis that for many diseases is inaccurate; there are frequently nodes of divergence and convergence of events to suggest a web or network of causation is a more apt term. For example, “congestive heart failure” has secondary consequences in other organ systems due to sluggish, compromised blood flow, reduced oxygen delivery to tissues, and compensatory (and eventu- ally decompensatory) hemodynamic changes—an example of diver- gence. Many causes of chronic lung injury ultimately manifest as a common histopathological picture of interstitial pneumonitis—an example of convergence. Diseases with a common ultimate cause, e.g., an inherited genetic mutation, can manifest differently even in identical twins, highlighting the crucial interaction between genes, environment, and other unknowns (epigenetics, serendipity) that contribute to the heterogeneous nature of nearly all diseases. Within and between categories of disease, two additional con- cepts deserve mention, lumping and splitting. Lumping occurs when distinct diseases are shown to share a common or related pathogenesis or therapeutic vulnerability. There are several recent notable examples of lumping: growth of two distinct types of cancer, chronic myeloid leukemia (CML) and gastrointestinal stromal tumor (GIST), is driven by overactivity of a shared intracel- lular growth promoting protein kinase cascade, due to mutations in related receptors that are susceptible to common kinase Histopathology: A Canvas and Landscape of Disease in Drug and Diagnostic Development 11 inhibitors [16]. Splitting occurs when a disease that appears homogeneous by traditional (often histopathological) criteria but manifests heterogeneously in a diverse population is subdivided into discrete diagnostic categories, typically by insights gained from new technologies. One example of splitting is a type of cancer termed diffuse large B-cell lymphoma (DLBCL). Although a given patient’s prognosis with DLBCL bears some relationship to tradi- tional, clinically measurable attributes such as tumor size, extent of spread throughout the body, and symptoms, most DLBCLs are indistinguishable from one another by histopathology: they consist of abnormal appearing lymphoid tumor cells mixed with host inflammatory cells. Expression profilingof mRNAs from a large set of DLBCL tumor tissues revealed at least two subtypes with mRNA expression profiles that bear some resemblance to benign B lymphocytes at different developmental stages—the germinal B-cell (GBC) type and activated B-cell (ABC) type [17]. Splitting is important, because the GBC and ABC tumor types probably have distinct ultimate causes (i.e., “driver” events such as gene mutations that give rise to, or propagate, the disease), prognoses, and, pre- sumably, therapeutic vulnerabilities that are under investigation. The right degree of splitting for a given disease is the essence of “personalized medicine.” Events that occur on a particular space or time scale in disease chain of causation can influence events that occur at vastly different space or time scales. Consequently, understanding chain of causa- tion can be a multidisciplinary pursuit, spanning epidemiology and public health to molecular biophysics and electrophysiology. The “channelopathies,” a heterogeneous group of diseases due to dysfunction of a variety of ion channels, are excellent examples of this principle [18]. Genetic mutation or acquired alteration in the hERG potassium channel subunit alters cardiac conduction that can manifest as chronically inefficient pumping of blood, or, in rare cases, sudden death [19]. Another class of diseases, character- ized by alterations in extracellular matrix proteins such as fibrillin, causes give rise to mechanical fragility of blood vessels at sites of high shear stress with loss of vascular tone, followed by high blood pressure and the often lethal consequence of aortic dissection [20]. Feedback can be important to understand chain of causation in disease. Feedback normally acts at critical upstream or downstream “nodes” (control points) to amplify or dampen the activity of biological systems. Feedback maintains homeostasis and reversibly adapts a system to acute, repetitive, and chronic perturbations. Proteins, molecular machines and pathways, cells, organ systems, and our bodies as a whole have evolved a variety of homeostatic mechanisms to optimize physiology as we interact with our envi- ronment through our life cycle. Enzyme production and activity are modified in response to metabolic demand; inflammation escalates upon pathogen contact and then regresses upon clearance of the 12 Keith A. Wharton Jr. invader; and blood pressure rises with arousal then falls with perceived safety—all examples of feedback. Some types of feedback occur within seconds, others over years. In disease, altered feedback can be considered an ultimate and/or proximate cause of disease, and can lead to specific signs and symptoms. Understanding how feedback mechanisms are altered in disease can be crucial to devel- oping effective targeting strategies, anticipating mechanisms of resistance to therapies, and managing side effects. Another concept widely invoked in normal physiology and disease is crosstalk: how one defined molecular pathway influences the activity of other pathways. While crosstalk in electronics is generally undesirable, crosstalk between biological pathways enables system integration. Altered inputs and outputs of different molecular pathways are features of many diseases, leading to altera- tions in cell function, tissue composition, and organ physiology, and so opposition of pathway alterations is a common strategy to target disease. Metabolic pathways that provide fundamental nutri- ents such as glucose and fatty and amino acids to cells and tissues of the body are among the most “crosstalked” pathways, in part because so many factors must be considered when allocating cellu- lar resources to energy utilization vs. storage. Diabetes is an exam- ple of a disease with altered feedback and crosstalk. In its common forms, it is characterized by failure to regulate serum glucose due to a deficit of, or altered responses to, insulin. Multiple inputs regulate insulin production and release from beta cells in the pancreatic islets, and the effect of circulating insulin in different cell types depends on inputs from other pathways. Crosstalk can be mediated by a direct physical interaction between two components of simul- taneously active yet distinct signaling pathways, and the conse- quences of the interaction can be inhibitory or synergistic. Understanding at what “level” crosstalk between two pathways occurs in health or diseases, including the cell types and subcellular compartments where key interactions occur, can inform diagnostic and therapeutic strategies. 8 The Significance of the Lesion If we must identify abnormalities in cells or their products to declare “disease,” then it becomes necessary to pinpoint, visualize, describe, and eventually measure the abnormality, known as the lesion. In pathology circles, the lesion is a focus of thought about disease. Intuitively, a lesion is mass or lump visible to the naked eye, but it can also be microscopic (i.e., visible only with a microscope) or molecular (e.g., a gene mutation is a genetic “lesion”). In toxicology, the lesion is the histopathological change in tissue structure and cell composition induced by exogenous agents, and whether it is reversible or not—i.e., whether it resolves a suitable Histopathology: A Canvas and Landscape of Disease in Drug and Diagnostic Development 13 time after the drug is withdrawn—often predicts whether a drug can be safely administered to humans. Until we have “tricorders” (a la Star Trek) capable of disease diagnosis at a distance, in most cases it is only possible to study the nature of lesions in live patients by studying diseased tissue, a fluid sample containing analytes (molecules that can be identified and measured) that originated from the diseased tissue, or another tissue that is specifically altered as a consequence of the diseased cells or tissues. Beyond routine histopathologic examination, attempts to understand lesions in diseased tissue must pay strict attention to tissue procurement, handling, and processing in order to preserve any molecules of interest (i.e., the analytes to be measured). Lesions, or, properly stated, visual representations of lesions, can be monitored in live patients by a variety of imaging modalities including CT, MRI, PET, SPECT, etc. Identifying and characterizing culprit lesions in disease are key to understand chain of causation/pathogenesis as well as to devise therapeutic strategies. Although a lesion can be a variety of sizes, Virchow—the “Father of Microscopic Pathology” who advocated for microscopy in disease diagnosis—espouses the central role of the cell in disease pathogenesis in his nineteenth century Pathology text: . . .the chief point in this application of histology to pathology is to obtain recognition of the fact, that the cell is really the ultimate morphological element in which there is any manifestation of life, and we must not transfer the seat of action to any point beyond the cell. [21] Virchow’s quote is simultaneously prescient and timeless: he implies efforts to understand each disease should focus on the structure and function of cells. Thus, for each disease, the key questions become: Which cells are diseased? What abnormalities exist within the diseased cells? How do diseased cells affect nearby cells to create the manifestations of disease? How can we detect the disease with a diagnostic test? How might we design therapies that reverse or mitigate disease-associated abnormalities in diseased tissues (efficacy) with acceptable consequences for nondiseased tissues (safety/ toxicity)? 9 Heterogeneity of Disease Medical textbooks impart the illusion to students (and the lay public with unlimited internet access) that diseases, as they com- mence in patients, tidily fit into categories. Experienced clinicians know better, attesting to the unpredictable and heterogeneous
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